The present invention relates to a process for producing a recombinant protein, in particular a process for producing a recombinant protein comprising growing a host cell expressing the recombinant protein and a step of maturing the host cell. The invention also provides for a recombinant protein obtainable by the process of the invention and immunogenic compositions or vaccines comprising the recombinant protein.
Expression of certain toxins is known to be challenging, for example Diphtheria toxin. DT may be produced by purification of the toxin from a culture of Corynebacterium diphtheriae followed by chemical detoxification, or may be made by purification of a recombinant, or genetically detoxified analogue of the toxin (for example CRM197, or other mutants as described in U.S. Pat. No. 4,709,107, U.S. Pat. No. 5,846,711, U.S. Pat. No. 5,601,827, and U.S. Pat. No. 5,917,017).
Production of significant quantities of diphtheria toxins such as CRM197 for use in vaccines has been hindered due to low protein abundance. This problem has been addressed previously by expressing CRM197 in E. coli (Bishai et al., J. Bacteriology 169:5140-5151 (1987). Bishai et al. describe the expression of a recombinant fusion protein containing diphtheria toxin (including the tox signal sequence) this led to the production of degraded protein.
Cloning of Diphtheria fragments containing the tox signal sequence and expression of these sequences in Escherichia coli involves certain difficulties. The expressed protein is secreted into the periplasmic space and this secretion is associated with decreased viability of the host cells (O'Keefe et al., Proc. Natl. Acad. Sci., 86:343-346 (1989)) and increased proteolysis of the recombinant protein (Bishai et al., J. Bacteriology 169:5140-5151 (1987). For these reasons removal of the tox signal sequence so that expression is no longer periplasmic has been suggested, this can increase expression of Diphtheria toxoids (Bishai et al).
PCT/EP2010/065047 (WO 2011/042516) discloses, for the first time, successful periplasmic expression of CRM197. This increases the yield of CRM197, however even here improvements to the extraction process can be made to increase the yield. Rathore discloses the optimization of an osmotic shock procedure for isolation of a protein product expressed in E. coli (Rathore et al Biotechnol. Prog. 2003, 19, 1541-1546). Bochner et al also discloses a method for recovering periplasmic protein from a host cell (U.S. Pat. No. 4,680,262).
Thus the present invention provides an improved process for production of a recombinant polypeptide comprising a step of maturing the host cell, wherein this step may comprise any one or more of the following:                (1) subjecting the host cell to a pH shock;        (2) incubating the host cell; or        (3) freezing the host cell.        
This step of maturing the host cell has the surprising result of substantially increasing the efficiency of protein extraction.